Pharmaceutical preparation and method of treatment of human malignancies with arginine deprivation

ABSTRACT

The present invention provides an isolated and substantially purified recombinant human arginase having sufficiently high enzymatic activity and stability to maintain Adequate Arginine Depletion in a patient. The present invention also provides a pharmaceutical composition comprising the modified invention enzyme and method for treatment of diseases using the pharmaceutical composition.

FIELD OF INVENTION

The present invention is related to pharmaceutical compositionscontaining arginase and use therefor. In particular, the presentinvention is related to pharmaceutical compositions that have thecapability of reducing the arginine level in patients with tumours andits use for treatment of human malignancies. The present invention alsorelates to a method of producing a recombinant protein.

BACKGROUND OF INVENTION

Arginase I (EC 3.5.3.1; L-arginine amidinohydrolase), is a key mammalianliver enzyme that catalyses, the final step in the urea formation in theUrea cycle, converting arginine into ornithine and urea. Rat liverextract, which has a high content of arginase, was found to haveanti-tumour properties in vitro when it was accidentally added to tumourcell culture medium (Burton et al., 1967, Cytolytic action ofcorticosteroids on thymus and lymphoma cells in vitro. Can J. Biochem.45, 289-297). Subsequent experiments showed that the anti-tumourproperties of the enzyme were due to depletion of arginine, which is anessential amino acid in the culture medium. At below 8 μM levels ofarginine, irreparable cell death in cancer cells occurred (Storr &Burton, 1974, The effects of arginine deficiency on lymphoma cells. Br.J. Cancer 30, 50-59).

A more novel aspect of arginine centers on its role as the directprecursor for the synthesis of the potent signalling molecule nitricoxide (NO), which functions as a neurotransmitter, smooth musclerelaxant, and vasodilator. Biosynthesis of NO involves a Ca⁺⁺,NADPH-dependent reaction catalysed by nitric oxide synthase (NOS).Another recognized role of arginine is that it acts as a precursor, viaornithine, of the polyamines, spermidine and spermine, which participatein diverse physiologic processes including cell proliferation and growth(Wu & Morris, 1998, Arginine metabolism: nitric oxide and beyond.Biochem. J. 336, 1-17).

Arginine also serves as a substrate for several important enzymes,including nitric oxide synthase (NOS). There are three types of NOSs,nNOS, eNOS and iNOS, all convert arginine to nitric oxide andcitrulline. The facial flushes induced by NO, for instance, is mediatedthrough nNOS, the neuronal type of NOS. iNOS, the inducible NOS isproduced by macrophages and the NO so produced from arginine duringsepticaemia causes vasodilation in endotoxic shock. eNOS, theendothelial NOS, is produced by endothelial cells in blood vessels. Itconverts arginine into NO, which then causes de-aggregation of plateletsin the endothelial surfaces through cGMP mechanism. NO produced fromeNOS in the local endQthelial lining has a half-life of about 5 secondsand diffusion distance of about 2 microns.

The productions of these enzymes are controlled by different NOS genes(NOS1, NOS2, NOS3) encoded in chromosomes 12, 17 & 7, respectively.These genes share strikingly similar genomic structures in size of exonsand the location of the splice junctions.

The in vitro anti-tumour activities of arginine depletion were confirmedrecently by a group in Scotland, UK (Scott et al., 2000, Single aminoacid (arginine) deprivation: rapid and selective death of culturedtransformed and malignanat cells. Br. J. Cancer 83, 800-810; Wheatley etal., 2000, Single amino acid (arginine) restriction: Growth and Death ofcultured HeLa and Human Diploid Fibroblasts. Cellular Physiol. Biochem.10, 37-55). Of the 24 different tumour cell lines tested, which includedcommon cancers such as breast, colorectal, lung, prostate and ovaries,all died within 5 days of arginine depletion. Using flow-cytometrystudies, the group was able to show that normal cell lines would enterinto quiescence for up to several weeks in G0 phase of the cell cyclewithout any apparent harm. Tumour cells, however, would proceed pass the“R” point in the G1 phase and enter the S phase with deficiency ofarginine. Without arginine, which is an irreplaceable amino acid,protein synthesis is deranged. Some cell lines were shown to die fromapoptosis. More excitingly, repeated depletions can bringforth tumourkill without “resistance” being developed (Lamb et al., 2000, Singleamino acid (arginine) deprivation induces G1 arrest associated withinhibition of Cdk4 expression in cultured human diploid fibroblasts.Experimental Cell Research 225, 238-249).

Despite the promising in vitro data, attempts with arginine depletion totreat cancer in vivo were unsuccessful. The original Storr groupattempted to treat tumour-bearing rats with intraperitoneal liverextracts and met with no success (Storr & Burton, 1974, The effects ofarginine deficiency on lymphoma cells. Br. J. Cancer 30, 50-59). It isnow generally recognized that under normal physiological condition, theblood plasma arginine level and indeed that of other amino acids too,are kept between the normal ranges (100-120 μM) with muscle being themain regulator. In the face of amino acid deficiency, intracellularprotein breakdown pathways are activated (proteasomal and lysosomal)releasing amino acids into the circulation (Malumbres & Barbacid, 2001,To cycle or not to cycle: a critical decision in cancer. Nature Reviews,1, 222-231). This amino acid homeostatic mechanism keeps the variousamino acid levels at constant ranges. Thus, previous attempts to depletearginine with various physical methods or arginine degrading enzymeshave failed because of the body's amino acid homeostatic mechanism.

To overcome the problem on the body's natural homeostatic tendencies,Tepic et al. in U.S. Pat. No. 6,261,557 described a therapeuticcomposition and method for treatment of cancer in which an argininedecomposing enzyme is used in combination with a protein breakdowninhibitors such as insulin in order to prevent the muscles of the bodyfrom replenishing the depleted arginine.

Although insulin can act as a protein breakdown inhibitor, it also hasfar-reaching physiological effects on the human body that may causefatal problems if blood glucose levels of the patient are not strictlymaintained within the narrow normal range. It is therefore an object tothe present invention to find improved method of treatment andcompositions for the treatment of cancer.

SUMMARY OF INVENTION

Accordingly, the present invention provides, in one aspect, an isolatedand substantially purified recombinant human arginase I (hereinafterreferred to as “Arginase” for ease of description unless otherwisestated) having a purity of 80-100%. In the preferred embodiment, theArginase has a purity of between 90-100%. In the most preferredembodiment, the Arginase according to the present invention is at least99% pure. In the example described below, the Arginase is more than99.9% pure based on densitometry tracing after SDS-PAGE separation. Inanother preferred embodiment, the Arginase of the present invention ismodified to have sufficiently high enzymatic activity and stability tomaintain “adequate arginine deprivation” (hereinafter referred to as“AAD”) in a patient for at least 3 days. One preferred method ofmodification is an amino-terminal tag of six histidines. Anotherpreferred modification is pegylation to increase the stability of theenzyme and minimise immunoreactivity illicited by the patient thereto.In the example described below, the Arginase has a plasma 12-life of atleast about 3 days and specific activity of at least about 250 I.U./mg.

In another aspect of the present invention, a method is provided forproducing a recombinant protein comprising the steps of (a) cloning agene encoding the protein; (b) constructing a recombinant Bacillussubtilis strain for expression of said protein (c) fermenting saidrecombinant B. subtilis cells using fed-batch fermentation; (d)heat-shocking said recombinant B. subtilis cells to stimulate expressionof said recombinant protein; and (e) purifying said recombinant proteinfrom the product of said fermentation. In the preferred embodiment, aprophage is used as the recombinant strain. Using the fed-batch methodof fermentation and prophage described above for the cloning andexpression of human recombinant arginase, there is more than a 4-foldincrease in maximum optical density at wavelength of 600 nm (OD)reached, and more than 5 times improvement in both the yield andproductivity of the Arginase as shown in Example 3 in the next section.In a further embodiment, the fermenting step can be scaled up forproducing the recombinant protein. In a further embodiment, thefermenting step is performed using a well-defined feeding medium of180-320 g/L glucose, 2-4 g/L MgSO₄.7H₂O, 45-80 g/L tryptone, 7-12 g/LK₂HPO₄ and 3-6 g/L KH₂PO₄. The use of a well-defined medium preventsundesirable material from being purified together with the recombinantprotein, making the method safe and efficient for the production ofpharmaceutical grade recombinant material.

In yet another preferred embodiment, the human Arginase gene is providedwith an additional coding region that encodes six additional histidinesat the amino-terminal end thereof, and the purifying step comprises achelating column chromatography step. In a further preferred embodiment,the Arginase enzyme is further modified by pegylation to improvestability.

In another aspect of the present invention, there are further providedpharmaceutical compositions comprising Arginase. In the preferredembodiment, the Arginase has sufficiently high enzymatic activity andstability to maintain AAD in a patient for at least 3 days. In the mostpreferred embodiment, the Arginase is further modified by pegylation toimprove stability and minimise immunoreactivity.

According to another aspect of the present invention, a pharmaceuticalcomposition is further formulated using Arginase.

In yet another aspect of the present invention, a method for treatmentof a disease is provided comprising administering a formulatedpharmaceutical composition of the present invention to a patient tomaintain the arginine level in such a patient to below 10 LM for atleast 3 days without the need for other protein breakdown inhibitors. Inone of the preferred embodiments, no insulin is administered exogenouslyfor non-diabetic patients.

Furthermore, the most preferred treatment method of the presentinvention involves the monitoring of the patient's blood for plateletcount (preferably maintained above 50,000×10⁹) and prothrombin time(maintained no more than 2 times normal). No nitric oxide producer isexogenously administered unless these levels of platelet count andprothrombin time are not reached.

In another preferred embodiment of this aspect of the present invention,pegylated Arginase is given as short infusion of over 30 minutes at3,000-5,000 I.U./kg in short infusion. arginine levels and Arginaseactivity are taken before Arginase infusion and daily thereafter. If AADis not achieved on day 2, the dose of the next infusion of Arginase isunder the discretion of the treating physician. The maximum toleratedduration of AAD is defined as the period of time during which bloodpressure is under control (with or without medication as deemappropriate by the treating physician), platelet count above 50,000×10⁹and prothrombin time less than 2× normal. As with arginine levels,complete blood count (CBC) and prothrombin time (PT) are taken daily.Liver chemistry is monitored at least twice weekly during the treatment.

The experimental data provided in the following detailed descriptionshows that arginase, if provided at sufficiently potent form, is usefulfor the treatment of maligancies. Although recombinant human arginase Iis the specific embodiment of an arginase that is used for the presentdisclosure, it is clear that other forms of arginase and/or from othersources may be used in accordance with the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows plasmid map of pAB101. This plasmid carries the geneencoding Arginase (arg) and only replicates in E. coli but not in B.subtilis.

FIGS. 2A, 2B and 2C show nucleotide sequence and its deduced amino acidsequence of the human Arginase I. FIG. 2A shows the nucleotide sequence(SEQ ID NO: 1) from EcoRI/MunI to XbaI sites of plasmid pAB101.Nucleotide (nt) 1-6, EcoRI/MunI site; nt 481-486, -35 region of promoter1; nt 504-509, -10 region of promoter 1; nt 544-549, -35 region ofpromoter 2; nt 566-571, -10 region of promoter 2; nt 600-605, ribosomebinding site; nt 614-616, start codon; nt 632-637, NdeI site; nt1601-1603, stop codon; nt 1997-2002, XbaI site.

FIG. 2B shows the encoding nucleotide sequence (SEQ ID NO: 2) and itscorresponding encoded amino acid sequence (SEQ ID NO: 3) of a modifiedhuman Arginase. Nucleotide 614-1603 from FIG. 2A is a encoding regionfor the amino acid sequence of the modified Arginase. The 6×His (SEQ IDNO: 4) tag at the N-terminus is underlined. Translation stop codon isindicated by asterisk.

FIG. 2C shows the encoding nucleotide sequence (SEQ ID NO:8) and itscorresponding encoded amino acid sequence (SEQ ID NO:9) of the normalhuman Arginase I.

FIG. 3 is a schematic drawing of the construction of a B. subtilisprophage allowing expression of Arginase.

FIGS. 4A and 4B show the time-course for fermentation in a 2-literfermentor by the recombinant Bacillus subtilis strain LLC101. FIG. 4Ashows the results obtained from the batch fermentation. FIG. 4B showsthe results obtained from the fed-batch fermentation.

FIGS. 5A and 5B show history plots of the fermentation showing thechanges of parameters such as temperature, stirring speed, pH anddissolved oxygen values. FIG. 5A shows the history plot from the batchfermentation. FIG. 5B shows the history plot from the fed-batchfermentation.

FIGS. 6A and 6B show the results of biochemical purification of humanArginase at 3 h after heat shock by the first 5-ml HiTrap Chelatingcolumn. FIG. 6A shows the FPLC running parameters and protein elutionprofile. FIG. 6B shows the SDS-PAGE (12%) analysis of 5 μl of each ofthe fractions 11-31 collected from the column. The protein gel wasstained with coomassie brilliant blue and destained to show the proteinbands. Lane M: low-range molecular weight marker (1 μg per band;Bio-Rad), with MW (Daltons): 97,400; 66,200; 45,000; 31,000; 21,500;14,400.

FIGS. 7A and 7B show results of purification of the human Arginase at 3h after heat shock by the second 5-ml HiTrap Chelating column. FIG. 7Ashows the FPLC running parameters and protein elution profile. FIG. 7Bshows the SDS-PAGE (12%) analysis of 1 μl of each of the fractions 9-39collected from the column. The protein gel was stained with coomassiebrilliant blue and destained to show the protein bands. Lane M:low-range molecular weight marker (1 μg per band; Bio-Rad), with MW(Daltons): 97,400; 66,200; 45,000; 31,000; 21,500; 14,400.

FIGS. 8A and 8B show results of purification of the human Arginase at 6h after heat shock by the first 5-ml HiTrap Chelating column. FIG. 8Ashows FPLC running parameters and protein elution profile. FIG. 8B showsthe SDS-PAGE (12%) analysis of 2.5 μl of each of the fractions 10-32collected from the column. The protein gel was stained with coomassiebrilliant blue and destained to show the protein bands. Lane M:low-range molecular weight marker (1 μg per band; Bio-Rad), with MW(Daltons): 97,400; 66,200; 45,000; 31,000; 21,500; 14,400.

FIGS. 9A and 9B show results of purification of the human Arginase at 6h after heat shock by the second 5-ml HiTrap Chelating column. FIG. 9Ashows FPLC runing parameters and protein elution profile. FIG. 9B showsthe SDS-PAGE (12%) analysis of 2 μl of each of the fractions 8-E6collected from the column. The protein gel was stained with coomassiebrilliant blue and destained to show the protein bands. Lane M:low-range molecular weight marker (1 μg per band; Bio-Rad), with MW(Daltons): 97,400; 66,200; 45,000; 31,000; 21,500; 14,40.

FIG. 10 shows the time-course of bacterial cell growth when heat shockwas performed at a higher cell density. Heat shock was performed at 8 hwhen the culture density (OD_(600nm)) was about 25.

FIG. 11 is the history plot of the fed-batch fermentation when heatshock was performed at a higher cell density. This plot shows thechanges of parameters such as temperature, stiring speed, pH anddissolved oxygen values.

FIGS. 12A and 12B show the results of purification of the human Arginaseat 6 h after heat shock (at a higher cell density of OD 25) by the first5-ml HiTrap Chelating column. FIG. 12A shows FPLC running parameters andprotein elution profile. FIG. 12B shows results of SDS-PAGE (12%)analysis of 5 μl of each of the fractions 16-45 collected from thecolumn. The protein gel was stained with coomassie brilliant blue anddestained to show the protein bands. Lane M: low-range molecular weightmarker (1 μg per band; Bio-Rad), with MW (Daltons): 97,400; 66,200;45,000; 31,000; 21,500; 14,400. Lane “crude”: 5 μl of the crude cellextract before loading the column.

FIGS. 13A and 13B show the results of purification of the human Arginaseat 6 h after heat shock (at a higher cell density of OD 25) by thesecond 5-ml HiTrap Chelating column. FIG. 13A shows FPLC runningparameters and protein elution profile. FIG. 13B shows the SDS-PAGE(12%) analysis of 5 μl of each of the fractions 7-34 collected from thecolumn. The protein gel was stained with coomassie brilliant blue anddestained to show the protein bands. Lane M: low-range molecular weightmarker (1 μg per band; Bio-Rad), with MW (Daltons): 97,400; 66,200;45,000; 31,000; 21,500; 14,400.

FIGS. 14A and 14B show the results of purification of the human Arginaseat 6 h after heat shock (at a higher cell density of OD 25) by the first1-ml HiTrap SP FF column. FIG. 14A shows FPLC running parameters andprotein elution profile. FIG. 14B shows the SDS-PAGE (12%) analysis of 5μl of each of the fractions A11-B7 collected from the column. Theprotein gel was stained with coomassie brilliant blue and destained toshow the protein bands. Lane M: low-range molecular weight marker (1 μgper band; Bio-Rad), with MW (Daltons): 97,400; 66,200; 45,000; 31,000;21,500; 14,400.

FIGS. 15A and 15B show the purification of the human Arginase at 6 hafter heat shock (at a higher cell density of OD 25) by the second 1-mlHiTrap SP FF column. FIG. 15A shows the FPLC running parameters andprotein elution profile. FIG. 15B shows the SDS-PAGE (12%) analysis of 5μl of each of the fractions A6-B12 collected from the column. Theprotein gel was stained with coomassie brilliant blue and destained toshow the protein bands. Lane M: low-range molecular weight marker (1 μgper band; Bio-Rad), with MW (Daltons): 97,400; 66,200; 45,000; 31,000;21,500; 14,400.

FIGS. 16A and 16B are the SDS-PAGE (15%) analysis of the human Arginasemodified with MnPEG-SPA (MW 5,000) using the Arginase:PEG mole ratio of1:50. FIG. 16A shows the results when reactions were performed on ice.Lane 1: low-range protein marker; Lane 2: Arginase (5.35 μg) without PEGadded (control); Lane 3: 1 h after reaction; Lane 4: 0.5 h afterreaction; Lane 5: 2 h after reaction; Lane 6: 3 h after reaction; Lane7: 4 h after reaction; Lane 8: 5 h after reaction; Lane 9: 23 h afterreaction. FIG. 16B shows the results when reactions were performed atroom temperature. Lane 1: low-range protein marker, Lane 2: Arginase(5.35 μg) without PEG added (control); Lane 3: 1 h after reaction; Lane4: 0.5 h after reaction; Lane 5: 2-h after reaction; Lane 6: 3 h afterreaction; Lane 7: 4 h after reaction; Lane 8: 5 h after reaction; Lane9: 23 h after reaction.

FIGS. 17A and 17B are the SDS-PAGE (15%) analysis of the human Arginasemodified with mPEG-SPA (MW 5,000) using the Arginase:PEG mole ratio of1:20. FIG. 17A shows the results when reactions were performed on ice.Lane 1: low-range protein marker; Lane 2: Arginase (5.35 μg) without PEGadded (control); Lane 3: 1 h after reaction; Lane 4: 0.5 h afterreaction; Lane 5: 2 h after reaction; Lane 6: 3 h after reaction; Lane7: 4 h after reaction; Lane 8: 5 h after reaction; Lane 9: 23 h afterreaction. FIG. 17B shows the results when reactions were performed atroom temperature. Lane 1: low-range protein marker; Lane 2: Arginase(5.35 μg) without PEG added (control); Lane 3: 1 h after reaction; Lane4: 0.5 h after reaction; Lane 5: 2 h after reaction; Lane 6: 3 h afterreaction; Lane 7: 4 h after reaction; Lane 8: 5 h after reaction; Lane9: 23 h after reaction.

FIG. 18A is the SDS-PAGE (15%) analysis of the human Arginase modifiedwith mPEG-CC (MW 5,000). The reactions were performed on ice. Lane 1:low-range protein marker; Lane 2: Arginase (5.35 μg) without PEG added(control); Lane 3: 2 h after reaction with Arginase:PEG mole ratio of1:50; Lane 4: empty; Lane 5: 23 h after reaction with Arginase:PEG moleratio of 1:50; Lane 6: 2 h after reaction with Arginase:PEG mole ratioof 1:20; Lane 7: 5 h after reaction with Arginase:PEG mole ratio of1:20; Lane 8: 23 h after reaction with Arginase:PEG mole ratio of 1:20.

FIG. 18B shows the SDS-PAGE (12%) analysis of the native and thepegylated Arginase which are highly active and stable. Lane 1: Low-rangeprotein marker (Dio-rad); Lane 2: Native Arginase (1 μg); Lane 3:Pegylated Arginase (1 μg); Lane 4: Pegylated Arginase afterultra-dialysis (1.5 μg).

FIGS. 19A and 19B show the measurement of the isolated recombinant humanArginase purity. FIG. 19A shows that for Lane 1: 5 μg of purified E.coli-expressed recombinant human Arginase obtained from methodsdescribed by Ikemoto et al. (Ikemoto et al., 1990, Biochem. J. 270,697-703). Lane 2: 5 μg of purified B. subtilis-expressed recombinanthuman Arginase obtained from methods described in this report. FIG. 19Bshows the analysis of densities of protein bands shown in FIG. 19A withthe Lumianalyst 32 program of Lumi-imagerm (Roche MolecularBiochemicals). Upper panel: results from lane 1 of FIG. 19A. Lowerpanel: results from lane 2 of FIG. 19A.

FIG. 20 is a diagram to show the stability of the pegylated Arginase invitro in human blood plasma.

FIGS. 21 and 22 show the half-life determination in vivo of pegylatedArginase obtained from the method described in example 8A. FIG. 21 showsthe in vivo activity of the pegylated Arginase produced according to thepresent invention using the activity test described in Example 9A.

FIG. 22 is a plot from which the first half-life and the secondhalf-life of the pegylated Arginase are determined.

FIG. 23 is a comparison of arginine depletion in four groups oflaboratory rats administered intraperitoneally with different dosages ofpegylated recombinant human Arginase (500 I.U., 1000 I.U., 1500 I.U.,and 3000 I.U.).

FIG. 24 shows the comparison of survival rate, average tumour size andtumour growth rate of tumours between 2 groups of nude mice which havetumours induced by implantation with Hep3B cells. One group was treatedwith Arginase with dosage of 500 I.U. intraperitoneally while the othercontrol group was not treated with Arginase.

FIGS. 25A and 25B show the comparison of average tumour size and averagetumour weight between 2 groups of nude mice which have tumours inducedby implantation with PLC/PRF/5 cells. One group was treated withArginase with dosage of 500 I.U. intraperitoneally while the othercontrol group was not treated with Arginase.

FIGS. 26A and 26B show the comparison of average tumour size and averagetumour weight of 2 groups of nude mice which have tumours induced byimplantation with HuH-7 cells. One group was treated with Arginase withdosage of 500 I.U. intraperitoneally while the other control group wasnot treated with Arginase.

FIG. 27 shows the comparison of average tumour size of 2 groups of nudemice which have tumours induced by implantation with MCF-7 cells. Onegroup was treated with Arginase with dosage of 500 I.U.intraperitoneally while the other control group was not treated withArginase.

FIG. 28 and FIG. 29 show in vivo arginine and CEA levels respectively ofthe patient during treatment as described in Example 12.

DETAILED DESCRIPTION

As used herein, the term “pegylated Arginase” refers to Arginase I ofpresent invention modified by pegylation to increase the stability ofthe enzyme and minimise immunoreactivity.

As used herein, the phrase “substantially the same”, whether used inreference to the nucleotide sequence of DNA, the ribonucleotide sequenceof RNA, or the amino acid sequence of protein, refers to sequences thathave slight and non-consequential sequence variations from the actualsequences disclosed herein. Species with sequences that aresubstantially the same are considered to be equivalent to the disclosedsequences and as such are within the scope of the appended claims. Inthis regard, “slight and non-consequential sequence variations” meansthat sequences that are substantially the same as the DNA, RNA, orproteins disclosed and/or claimed herein are functionally equivalent tothe sequences disclosed and/or claimed herein. Functionally equivalentsequences will function in substantially the same manner to producesubstantially the same compositions as the nucleic acid and amino acidcompositions disclosed and claimed herein. In particular, functionallyequivalent DNAs encode proteins that are the same as those disclosedherein or proteins that have conservative amino acid variations, such assubstitution of a non-polar residue for another non-polar residue or acharged residue for a similarly charged residue. These changes includethose recognized by those of skill in the art not to substantially alterthe tertiary structure of the protein. The term “sufficiently highenzymatic activity” refers to the enzyme specific activity of therecombinant human Arginase for at least 250 I.U./mg, preferably at least300-350 I.U./mg, more preferably at least 500 I.U./mg. In the preferredembodiment, the Arginase has a specific activity of 500-600 I.U./mg. Theterm “stability” refers to in vitro stability of the Arginase. Morepreferably, the stability refers to in vivo stability. The rate ofdecrease of enzyme activity is inversely proportional to the plasmastability of the isolated, purified recombinant human Arginase. Thehalf-life of such a human Arginase in plasma is calculated.

As used herein, the term “adequate arginine deprivation” (AAD) refers toin vivo arginine level at or below 10 μM. The term “disease” refers toany pathological conditions, including but not limited to liver diseasesand cancer.

As used herein, the term “half-life” (½-life) refers to the time thatwould be required for the concentration of the Arginase in human plasmain vitro, to fall by half. In early 2001, three cases of spontaneoustransient remission of hepatocellular carcinoma (HCC) were observed byone of the inventors of the present invention. All three patients hadspontaneous rupture of HCC with resulting haemoperitoneum. In one case,the plasma arginine was found to be as low as 3 μM and arginine level inthe ascitic fluid at 7 μM. These patients all had spontaneous remissionof their liver tumour with normalization of alpha-fetoprotein (AFP)after ruptured liver lesions in the absence of any treatment using anypharmaceutical drugs. One patient had remission of his HCC for over 6months. In accordance with the present invention, it is believed thatsuch prolonged remission is caused by arginine depletion due to thespontaneous and sustained release of endogenous Arginase into theperitoneum from the rupture of the liver. Thus, the inventors inferredthat prolonged arginine depletion was the causative factor leading toremission of HCC.

A series of experiments was then designed by the inventor of the presentinvention to show that endogenous hepatic Arginase can be released fromthe liver after transhepatic arterial embolisation causing systemicarginine deprivation. This has now been filed in the U.S. provisionalpatent application No. 60/351,816, which is incorporated by referenceherewith. In the experiments designed by the inventor, moderate andmeasurable amount of endogenous hepatic arginase was found to bereleased into the systemic circulation in patients with unresectablemetastatic HCC after hepatic arterial embolisation treatment usinglipiodol and gel foam that caused a temporary hepatic perfusion defect.High dose insulin infusion was incorporated into the treatment regime toinduce a state of hypoaminoacidemia. In a series of 6 cases of HCCtreated, 4 had extra hepatic remission of liver cancer suggesting thetreatment effects are systemic. One patient had sustained completeremission, both radiological with CT and PET in his liver andextrahepatic disease (celiac adenopathy). His AFP level dropped tonormal within 3 weeks and sustained for over 4 months. Interval CT at 4months showed no demonstrable tumour both hepatic or extrahepatic. Theother 3 patients all had remission of their extra hepatic disease (onepulmonary, one mesenteric/retroperitoneal/bone and one retroperitonealadenopathy) on PET scan at 4 weeks after embolisation. On testing theirArginase activities and arginine levels, all had adequate argininedepletion for a period of time lasting from 2 hours to 2 days. In factthe duration of AAD correlated well with the degree and duration ofremission of the tumour, both hepatic and extra-hepatic.

Although the transhepatic arterial embolisation technique was performedin conjunction with high doses of insulin infusion, the inventors, inaccordance with the present invention, subsequently came to therealisation that the need for the administration of insulin was due tothe fact that insufficient arginase activities may be released into thesystem of the patient such that any protein degradation from the musclewould have a compensatory effect from the arginine deprivation andrender the treatment ineffective. In accordance with the presentinvention, the inventors realised that in order to improve the treatmentand to eliminate the need for administration of insulin in conjunctionwith the arginine deprivation treatment, arginase activity has to bepresent in sufficiently high amounts in the patient's system in order tocounteract any protein degradation from the muscle. In accordance withthe present invention, the inventors therefore set out to produce anArginase enzyme that had sufficiently high enzymatic activities andstability to maintain “adequate arginine deprivation” hereinafterreferred to as “AAD”) of below 10 μM in the patient without the need toadministrate high dose of insulin. Thus, in addition to augmenting theendogenous Arginase, the highly stable and active Arginase according tothe present invention provides the additional benefit of allow AAD to beattained without the administration of a protein degradation inhibitor,which has undesirable side effects on the patient.

Systemic depletion of arginine may cause other undesirable side effectrelated to nitric oxide deficiency. These include hypertension due toabsence of vasodilator effect of NO on vascular endothelium, plateletaggregation and thrombocytopenia secondary to lack of NO and depletionof early clotting factors related to temporary cessation of celldivision. The inventor recognized, however, that in nitric oxide knockout mice the animals are not hypertensive and have normal lifeexpectance with normal platelet counts. Thus, in accordance with anotheraspect of the present invention and in patients with thrombocytopenia,no overt haemorrhagic tendency is seen until platelet count is wellbelow 50,000×10⁹. In patients with thrombotic tendency, therapy entailsprolonging the prothrombin time for up to 2× normal.

The following detailed examples teach how to make and use a highlystable and active Arginase according to the present invention. Example 1describes the construction of the recombinant strain of Bacillussubtilis LLC101 containing the human Arginase I gene. This is followedby two examples of fermentation of the recombinant B. subtilis. In theinitial fermentation experiments of the recombinant LLC101 cells, batchfermentation and fed-batch fermentation were conducted in a 2-Lfermentor. It was found that under batch conditions sufficiently highcell density could not be attained. Only under the fed-batch conditionsprovided in accordance with the present invention would cell density beincreased to above 10 OD (optical density). These experiments andresults are shown in Examples 2A and 2B. A comparison of the 2fermentation methods is shown in Example 3. Fed-batch fermentationoperation was thus chosen for production of isolated and purifiedrecombinant human Arginase. The fed-batch fermentation was scaled up ina 100-L fermentor. The experiments and results are shown in Example 2C.

The LLC101 strain is a heat sensitive strain that causes expression ofthe Arginase upon heat shock at 50° C. In the initial optimisationexperiments, the heat shock treatment was performed at varying celldensities to obtain the optimal conditions under which maximum Arginasewould be produced. Examples 5 and 6 describe the purification processand the yield of purified Arginase thus obtained of two differentfed-batch fermentation runs with heat shock at two different OD (opticaldensity at 600 nm), 12.8 and 25. The experimental data showed thatalthough all heat shocks were applied during the exponential growthphase of the LLC101, introduction of heat shock at a lower cell density,e.g., 12.8 OD, produced better results.

Conditions for maximum expression of Arginase after heat shock was alsooptimised by varying the time of harvest after heat shock. Example 4shows results from harvesting the cells three hours after heat shock andusing a fed-batch fermentation process.

Example 5 describes a purification of Arginase 6 hours after heat shockat a cell density of 12.8 OD. Example 6 describes the purification ofArginase 6 hours after heat shock at a higher cell density of 250D.Example 7 shows a comparison of the data to compare the yield of theArginase under various harvesting and purification conditions. Thesedata show that harvesting cells 6 h after heat shock at a lower celldensity of 12.8 produced a higher Arginase yield of 162 mg/L. TheArginase was modified to improve stability. Example 8A shows oneprotocol for the pegylation of the Arginase using cyanuric chloride (cc)as the cross-linker at an ratio of 1:140 (Arginase:PEG) mole ratio.Example 8B describes a different pegylation protocol in which a muchlower proportion of cross-linker is added into the reaction mixture withthe enzyme. Both cc and succinimide of propionic acid (SPA) were testedas cross-linker. Experimental results show that the method as describedin Example 8B using SPA provided a pegylated Arginase with a 12-life of3 days and a specific activity of approximately 255 I.U./mg as discussedin Examples 9 and 10. Example 8C describes a method for preparing ahighly active pegylated Arginase, which has a specific activity of about592 I.U./mg.

Using the method as described above, a highly stable and active Arginasehas been produced. It has sufficiently high activity and stability toallow treatment of patients without significant use of a proteindegradation inhibitor because any replenishment of arginine by themuscle would be quickly removed by the systemic Arginase. Thus, adequatearginine deprivation of below 10 μM can be achieved without high dosesof exogenously administered insulin. Various treatment protocols usingthe Arginase according to the present invention is described in Example11. Example 12 illustrates the clinical data of a patient administeredwith Arginase to further support the treatment protocol shown in Example11.

Examples 13 to 14 are two animal studies on rats investigating doseresponses and safety doses of the Arginase according to the presentinvention. Examples 15 to 18 are another series of animal studies onnude mice to investigate the responses of tumours induced by differenthuman cancer cell lines upon arginine depletion induced byadministration of modified Arginase.

All references cited above are incorporated by reference herein. Thepractice of the invention is exemplified in the following non-limitedExamples. The scope of the invention is defined solely by the appendedclaims, which are in no way limited by the content or scope of theExamples.

EXAMPLES Example 1 Construction of the Recombinant Strain LLC101

(a) Isolation of the Gene Encoding Human Arginase I

The gene sequence of human Arginase I was published in 1987 (Haraguchi,Y. et al., 1987, Proc. Natl. Acad. Sci. 84, 412-415) and primersdesigned therefrom. Polymerase chain reaction (PCR) was performed toisolate the gene encoding a human Arginase using the Expand HighFidelity PCR System Kit (Roche). Primers Arg1(5′-CCAAACCATATGAGCGCCAAGTCCAGAACCATA-3′) (SEQ ID NO: 5) and Arg2(5′-CCAAACTCTAGAATCACA=TTTGAATGACATGGACAC-3′) (SEQ ID NO: 6),respectively, were purchased from Genset Singapore Biotechnology PteLtd. Both primers have the same melting temperature (Tm) of 72 degree C.Primer Arg1 contains a NdeI restriction enzyme recognition site(underlined) and primer Arg2 contains a XbaI site (underlined).These twoprimers (final concentration 300 nM of each) were added to 5 μl of thehuman liver 5′-stretch plus cDNA library (Clontech) in a 0.2-mlmicro-tube. DNA polymerase (2.6 units, 0.75 μl), the fourdeoxyribonucleotides (4 μl of each; final concentration 200 μM of each)and reaction buffer (5 μl) and dH₂O (17.75 μl) were also added. PCR wasperformed using the following conditions: pre-PCR (94 degree C., 5 min),25 PCR cycles (94 degree C., 1 min; 57 degree C., 1 min; 72 degree C., 1min), post-PCR (72 degree C., 7 min). PCR product (5 μl) was analyzed ona 0.8% agarose gel and a single band of 1.4 kb was observed. This DNAfragment contains the gene encoding Arginase.

(b) Isolation of Plasmid pSG1113

Plasmid pSG1113, which is a derivative of plasmid pSG703 (Thomewell, S.J. et al., 1993, Gene, 133, 47-53), was isolated from the E. coli DH5aclone carrying pSG1113 by using the Wlizard Plus Minipreps DNAPurification System (Promega) following the manufacturer's instruction.This plasmid, which only replicates in E. coli but not in B. subtilis,was used as the vector for the subcloning of the Arginase gene.

(c) Subcloning the 1.4 kb PCR Product into Plasmid pSG1113 to FormPlasmid pAB101

The PCR product, prepared using the above protocol, was treated withrestriction endonucleases NdeI and XbaI (Promega) in a reaction mediumcomposed of 6 mM Tris-HCl (pH 7.9), 6 mM MgCl₂, 150 mM NaCl, 1 mM DTT at37 degree C. for 1.5 h. After completion of the treatment, the reactionmixture was subjected to agarose gel (0.8%) electrophoresis, and the 1.4kb DNA fragment was recovered from the gel by using the Qiaex II GelExtraction Kit (Qiagen). Separately, the plasmid pSG1113 was treatedwith the same restriction endonucleases in the same way. Aftercompletion of the treatment, the reaction mixture was subjected toagarose gel (0.8%) electrophoresis, and a DNA fragment having a size ofabout 3.5 kb was recovered from the gel. This DNA fragment was joined byusing T4 DNA ligase to the above 1.4 kb DNA fragment. The ligationmixture was used to transform E. coli XLI-Blue using the conventionalcalcium method (Sambrook, J. et al., Molecular Cloning, A LaboratoryManual, second edition, Cold Spring Harbor Laboratory Press, New York,1989) and plated on nutrient agar plate containing 100 μg/ml ampicillin.Colonies were screened for a plasmid with the appropriate insert byrestriction analysis. The plasmid constructed was designated pAB101(FIG. 1). OR1 is the E. coli origin of replication and bla is theampicillin resistant marker gene. DNA sequencing was performed withprimers Arg1 (SEQ ID NO: 5), Arg2 (SEQ ID NO:6) and Arg6(5′-CTCTGGCCATGCCAGGGTCCACCC-3′) (SEQ ID NO: 7) to confirm the identityof the gene encoding Arginase (FIG. 2).

(d) Construction of the Novel Recombinant B. subtilis Prophage StrainLLC101

The plasmid pAB101 was extracted and purified from the clone carryingthe pAB101 by using the Wizard Plus Minipreps DNA Purification System(Promega). In the plasmid pAB101 (FIG. 1), the Arginase gene (arg) wasflanked by the 0.6 kb MunI-NdeI p105 phage DNA fragment (labelled as“p105”) and the cat gene (FIG. 1 and FIG. 3). This plasmid DNA (1 μg)was used to transform competent B. subtilis 1A304(+105MU331) accordingto the known method (Anagnostopoulos C. and Spizizen J., 1961, J.Bacteriol. 81, 741-746). The B. subtilis strain 1A304(φ105MU331) wasobtained from J. Errington (Thornewell, S. et al., 1993, Gene 133,47-53). The strain was produced according to the publications byThornewell, S. et al., 1993, Gene 133, 47-53 and by Baillie, L. W. J. etal., 1998, FEMS Microbiol. Letters 163, 43-47, which are incorporatedherein in their entirety. Plasmid pAB101 (shown linearized in FIG. 3)was transformed into the B. subtilis strain 1A304 (φ105MU331) withselection for the CmR marker, and the transformants were screened for anErs phenotype. Such transformants should have arisen from adouble-crossover event, as shown in FIG. 3, placing transcription of theArginase gene (arg) under the control of the strong phage promoter(Leung and Erington, 1995, Gene 154, 1-6). The thick lines represent theprophage genome, broken lines the B. subtilis chromosome, and thin linesplasmid DNA. The genes are shown in FIG. 3 as shaded arrows pointing inthe direction of transcription and translation. Regions of homology arebounded by broken vertical lines and homologous recombination events by‘X’.

Fifty-two chloramphenicol resistant (CmR) colonies were obtained fromplating 600 μl of the transformed cells on an agar plate containingchloramphenicol (5 μg/ml). Ten of these colonies were selected randomlyand streaked onto an agar plate containing erythromycin (20 μg/ml) andone of these colonies did not grow, indicating that it was erythromycinsensitive (Ers). This chloramphenicol resistant but erythromycinsensitive colony was thus isolated and named as LLC101. In thechromosome of this newly constructed prophage strain, the erythromycinresistance gene (ermC) was replaced by the Arginase gene (arg) by adouble crossover event in a process of homologous recombination. The 0.6kb MunI-NdeI φ105 phage DNA fragment (labelled as “φ105”) and the catgene provided the homologous sequences for the recombination. In thisway, the Arginase gene was targeted to the expression site in theprophage DNA of B. subtilis 1A304(φ105MU331) and the Arginase gene wasput under the control of the strong thermoinducible promoter (Leung, Y.C. and Errington, J., 1995, Gene 154, 1-6).

Fermentation of B. subtilis LLC101 Cells

Example 2A Batch Fermentation in a 2-Liter Fermentor

The B. subtilis LLC101 strain is maintained on a Nutrient Agar (beefextract 1 g/L, peptone 10 g/L, NaCl 5 g/L and agar 20 g/L) plate,supplemented with 5 mg/L of chloramphenicol. To prepare the innoculumfor batch and fed-batch fermentation, a few colonies of theaforementioned strain were transferred from a freshly prepared NutrientAgar plate into two 1-L flasks, each containing 80 mL of fermentationmedium containing glucose 5 g/L, tryptone 10 g/L, yeast extract 3 g/L,sodium citrate 1 g/L, KH₂PO₄ 1.5 g/L, K₂HPO₄ 1.5 g/L, and (NH₄)₂SO₄ 3g/L. The bacterial cell culture was cultivated at 37° C. and pH 7.0 onan orbital shaker rotating at 250 r.p.m. The cultivation was terminatedwhen OD_(600nm) reached 5.5-6.0 at about 9-11 h growth time. Then the160-nL culture broth was introduced into the 2-L fermentor containing1440-mL fermentation medium (glucose 5 g/L, tryptone 10 g/L, yeastextract 3 g/L, sodium citrate 1 g/L, KH₂PO₄ 1.5 g/L, K₂HPO₄ 1.5 g/L, and(NH₄)₂SO₄ 3 g/L). The batch fermentation was carried out at atemperature of 37° C. The pH was controlled at 7.0 by adding sodiumhydroxide and hydrochloric acid. The dissolved oxygen concentration wascontrolled at 20% air saturation with the adjustment of stirring speed.Heat shock was performed at 3.25 h when the culture density (OD_(600nm))was about 3.9. During the heat shock, the temperature of the fermentorwas increased from 37 degree C. to 50 degree C. and then cooledimmediately to 37 degree C. The complete heating and cooling cycle tookabout 0.5 h. The OD of the culture reached a maximum of about 6.4 at 3.5h after heat shock. Cells were harvested for separation and purificationof Arginase at 6 h after heat shock. The aforementioned strain producedactive human Arginase in an amount of about 30 mg/L of the fermentationmedium at 6 h after heat shock. The time-course of the fermentation isplotted in FIG. 4A. The history plot of this batch fermentation showingthe changes of parameters such as temperature, stirring speed, pH anddissolved oxygen values is depicted in FIG. 5A.

Example 2B Fed-Batch Fermentation in a 2-Liter Fermentor

The Fed-batch fermentation was carried out at 37 degree C., pH 7.0 anddissolved oxygen 20% air saturation. The inoculation procedure wassimilar to that of the batch fermentation described in Example 2A.Initially, the growth medium was identical to that used in the batchfermentation described in Example 2A. The feeding medium contained 200g/L glucose, 2.5 g/L MgSO₄.7H₂O, 50 g/L tryptone, 7.5 g/L K₂HPO₄ and3.75 g/L KHzPO₄. The medium feeding rate was controlled with the pH-statcontrol strategy. In this strategy, the feeding rate was adjusted tocompensate the pH increase caused by glucose depletion. This controlstrategy was first implemented when the glucose concentration decreasedto a very low level at about 4.5-h fermentation time. If pH>7.1, 4 mL offeeding medium was introduced into the fermentor. Immediately after theaddition of glucose, the pH value would decrease below 7.1 rapidly.After approximate 10 min, when the glucose added was completely consumedby the bacterial cells, the pH value would increase to a value greaterthan 7.1, indicating that another 4 mL of feeding medium was due to beadded into the fermentor. Heat shock was performed at 5-6 h when theculture density (OD_(600nm)) was between 12.0 and 13.0. During the heatshock, the temperature of the fermentor was increased from 37 degree C.to 50 degree C. and then cooled immediately to 37 degree C. The completeheating and cooling cycle took about 0.5 h. Cells were harvested forseparation and purification of Arginase at 3 h and 6 h after heat shock.The aforementioned strain produced active human Arginase in an amount ofat least about 162 mg per L of the fermentation medium at 6 h after heatshock. The time-course of the fermentation is plotted in FIG. 4B. Thehistory plot of this fed-batch fermentation showing the changes ofparameters such as temperature, stirring speed, pH and dissolved oxygenvalues is indicated in FIG. 5B.

Example 2C Fed-Batch Fermentation in a 100-Liter Fermentor

The Fed-batch fermentation was scaled up in a 100-L fermentor. Thefermentation was carried out at 37 degree C., pH 7.0, dissolved oxygen20% air saturation. A 10% inoculum was used. Initially, the growthmedium was identical to that used in the batch fermentation described inExample 2A. The feeding medium contained 300 g/L glucose, 3.75 g/LMgSO₄.7H₂O, 75 g/L tryptone, 11.25 g/L K₂HPO₄ and 5.625 g/L KH₂PO₄. Themedium feeding rate was controlled with a pH-stat control strategysimilar to that used in the fed-batch fermentation described in Example2B. Heat shock was performed at about 7.5 h when the culture density(OD_(600nm)) was between 11.5 and 12.5. During the heat shock, thetemperature of the fermentor was increased from 37 degree C. to 50degree C., maintained at 50 degree C. for 7 s and then cooledimmediately to 37 degree C. The complete heating and cooling cycle tookabout 0.5 h. Cells were harvested for separation and purification ofArginase at 2 h and 4 h after heat shock. The aforementioned strainproduced active human Arginase in an amount of at least about 74 mg and124 mg per L of the fermentation medium at 2 h and 4 h, respectively,after heat shock. These data show that harvesting cells 4 h after heatshock produced a higher Arginase yield than harvesting cells 2 h afterheat shock in a 100-L fermentor.

Example 3 Comparison of Batch and Fed-Batch Fermentation

Table 1 below compares the results of batch and fed-batch fermentation.The comparison demonstrates that the fed-batch fermentation was muchsuperior to the batch operation in terms of culture OD, Arginase yieldand productivity. TABLE 1 Batch Fed-batch Fermentation Fermentation TheOD at the start of heat 3.9 12.8 shock Maximum OD reached 6.0 26.8Arginase Yield (mg/L) 30 162 Arginase Productivity (mg/L-h) 2.5 13.5

Example 4 Purification of Arginase at 3H After Heat Shock AfterFed-Batch Fermentation at Low Cell Density

Fed-batch fermentation in a 2-liter fermentor was performed as describedin Example 2B. The cell density of the fed-batch culture was monitoredat 30 or 60 min interval and the temperature of the culture raised to50° C. for heat shock at 5.5 hours after the fermentation started whenthe OD of the culture reached 12.8 (see FIG. 4B and FIG. 5B).

The cell culture (470 ml) collected at 3 h after heat shock wascentrifuged at 5,000 rpm for 20 min at 4 degree C. to pellet the cells.The wet weight of the cells was 15.1 g. The culture supernatant liquorwas discarded and the cell pellet was stored at −80 degree C. The cellsare stable at this temperature for a few days. To extract intracellularproteins, the cell pellet was resuspended in 140 ml solubilizationbuffer [50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 5 mM MlSO₄, lysozyme (75μg/ml)]. After incubation at 30 degree C. for 15 min, the mixture wassonicated for eight times, each time lasted for 10 s (the total time was80 s), at 2 min intervals using the Soniprep 150 Apparatus (MSE). About500 units of deoxyribonuclease I (Sigma D 4527) was added and themixture was incubated at 37 degree C. for 10 min to digest thechromosomal DNA. After centrifugation at 10,000 rpm for 20 min at 4degree C., the supernatant, containing the crude protein extract, wasassayed for the presence of the Arginase activity and analyzed bySDS-PAGE (Laemmli, 1970, Nature, 227, 680-685).

A 5-ml HiTrap Chelating column (Pharmacia) was equilibrated with 0.1 MNiCl₂ in dH₂O, for 5 column volumes. The crude protein extract (140 ml)was loaded onto the column. Elution was performed with a linear gradient(0-100%) at a flow rate of 5 ml/min for 15 column volumes under thefollowing conditions: Buffer A=start buffer [0.02 M sodium phosphatebuffer (pH 7.4), 0.5 M NaCl]; Buffer B=start buffer containing 0.5 Mimidazole. The elution profile is shown in FIG. 6A and the protein gelis shown in FIG. 6B. Fractions 13-20 were pooled (16 ml) and diluted tentimes with start buffer [0.02 M sodium phosphate buffer (pH 7.4), 0.5 MNaCl]. This was loaded onto a second 5-ml HiTrap Chelating column(Pharmacia), repeating the same procedure as above. The elution profileis shown in FIG. 7A and the protein gel is shown in FIG. 7B. Fractions12-30 containing Arginase were pooled (38 ml) and salt was removed usinga 50-ml HiPrep 26/10 desalting column (Pharmacia) with the followingconditions: flow rate=10 mVmin, buffer=10 mM Tris-HCl (pH 7.4) andlength of elution=1.5 column volume. The protein concentration wasmeasured by the method of Bradford (Bradford, M. M., 1976, Anal.Biochem., 72, 248-254). A total of 56.32 mg of Arginase was purifiedfrom 470 ml cell culture. The yield of purified Arginase was estimatedto be 119.8 mg/l cell culture or 3.73 mg/g wet cell weight.

Example 5 Purification of Arginase at 6H After Heat Shock AfterFed-Batch Fermentation at Low Cell Density

Fed-batch fermentation in a 2-liter fermentor was performed as describedin Example 4. The cell culture (650 ml) collected at 6 h after heatshock at OD 12.8 was centrifuged at 5,000 rpm for 20 min at 4 degree C.to pellet the cells. The wet weight of the cells was 24 g. The culturesupernatant liquor was discarded and the cell pellet was stored at −80°C. The cells are stable at this temperature for a few days. To extractintracellular proteins, the cell pellet was resuspended in 140 mlsolubilization buffer [50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 5 mM MnSO₄,lysozyme (75 μg/ml)]. After incubation at 30 degree C. for 15 min, themixture was sonicated for eight times, each time lasted for 10 s (thetotal time was 80 s), at 2 min intervals using the Soniprep 150Apparatus (MSE). About 500 units of deoxyribonuclease I (Sigma D 4527)was added and the mixture was incubated at 37 degree C. for 10 min todigest the chromosomal DNA. After centrifugation at 10,000 rpm for 20min at 4 degree C., the supernatant, containing the crude proteinextract, was assayed for the presence of the Arginase activity andanalyzed by SDS-PAGE (Laemmli, 1970, Nature, 227, 680-685).

A 5-ml HiTrap Chelating column (Pharmacia) was equilibrated with 0.1 MNiCl₂ in dH₂O, for 5 column volumes. The crude protein extract (140 ml)was loaded onto the column. Elution was performed with a linear gradient(0-100%) at a flow rate of 5 m/min for 15 column volumes under thefollowing conditions: Buffer A=start buffer [0.02 M sodium phosphatebuffer (pH 7.4), 0.5 M NaCl]; Buffer B=start buffer containing 0.5 Mimidazole. The elution profile is shown in FIG. 8A and the protein gelis shown in FIG. 8B. Fractions 13-24 were pooled (24 ml) and diluted tentimes with start buffer [0.02 M sodium phosphate buffer (pH 7.4), 0.5 MNaCl]. This was loaded onto a second 5-ml HiTrap Chelating column(Phalmacia), repeating the same procedure as above. The elution profileis shown in FIG. 9A and the protein gel is shown in FIG. 9B. Fractions12-24 containing Arginase were pooled (26 ml) and salt was removed usinga 50-ml HiPrep 26/10 desalting column (Pharmacia) with the followingconditions: flow rate=10 ml/min, buffer=10 mM Tris-HCl (pH 7.4) andlength of elution=1.5 column volume. The protein concentration wasmeasured by the method of Bradford (Bradford, M. M., 1976, Anal.Biochem., 72, 248-254). A total of 85.73 mg of Arginase was purifiedfrom 650 ml cell culture. The yield of purified Arginase was estimatedto be 132 mg/l cell culture or 3.57 mg/g wet cell weight

Example 6 Purification of Arginase at 6H After Heat Shock at a HigherCell Density

In this particular fed-batch fermentation, the process was similar tothe above example except that the heat shock was performed at 8 h whenthe culture density (OD_(600nm)) was about 25. During the heat shock,the temperature of the fermentor was increased from 37 degree C. to 50degree C. and then cooled immediately to 37 degree C. The completeheating and cooling cycle took about 0.5 h. A portion of the cellculture (760 ml) was harvested for separation and purification ofArginase at 6 h after heat shock. The time-course of bacterial cellgrowth in this fermentation is plotted in FIG. 10. The history plot ofthis fed-batch fermentation showing the changes of parameters such astemperature, stirring speed, pH and dissolved oxygen values is indicatedin FIG. 11.

The cell culture (760 ml) collected at 6 h after heat shock wascentrifuged at 5,000 rpm for 20 min at 4 degree C. to pellet the cells.The wet weight of the cells was 32 g. The culture supernatant liquor wasdiscarded and the cell pellet was stored at −80 degree C. The cells arestable at this temperature for a few days. To extract intracellularproteins, the cell pellet was resuspended in 280 ml solubilizationbuffer [50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 5 mM MnSO₄, lysozyme (75μg/ml)]. After incubation at 30 degree C. for 15 min, the mixture wassonicated for eight times, each time lasted for 10 s (the total time was80 s), at 2 min intervals using the Soniprep 150 Apparatus (MSE). About500 units of deoxyribonuclease I (Sigma D 4527) was added and themixture was incubated at 37 degree C. for 10 min to digest thechromosomal DNA. After centrifugation at 10,000 rpm for 20 min at 4degree C., the supernatant, containing the crude protein extract, wasassayed for the presence of the Arginase activity and analyzed bySDS-PAGE (Laemmli, 1970, Nature, 227, 680-685).

A 5-ml HiTrap Chelating column (Pharmacia) was equilibrated with 0.1 MNiCl₂ in dH₂O, for 5 column volumes. The crude protein extract (280 ml)was loaded onto the column. Elution was performed with a linear gradient(0-100%) at a flow rate of 5 ml/min for 15 column volumes under thefollowing conditions: Buffer A=start buffer [0.02 M sodium phosphatebuffer (pH 7.4), 0.5 M NaCl]; Buffer B=start buffer containing 0.5 Mimidazole. The elution profile is shown in FIG. 12A and the protein gelis shown in FIG. 12B. Fractions 17-31 were pooled (30 ml) and dilutedten times with start buffer [0.02 M sodium phosphate buffer (pH 7.4),0.5 M NaCl]. This was loaded onto a second 5-ml HiTrap Chelating column(Pharmacia), repeating the same procedure as above. The elution profileis shown in FIG. 13A and the protein gel is shown in FIG. 13B. Fractions10-20 containing Arginase were pooled (22 ml) and salt was removed usinga 50-ml HiPrep 26/10 desalting column (Pharmacia) with the followingconditions: flow rate=10 ml/min, buffer=10 mM Tris-HCl (pH 7.4) andlength of elution=1.5 column volume. The sample was then loaded onto a1-ml HiTrap SP FF column (Pharmacia). Elution was performed with thefollowing conditions: flow rate=1 ml/min, Buffer A=10 mM Tris-HCl (pH7.4), Buffer B=10 mM Tris-HCl (pH 7.4) containing 1 M NaCl, lineargradient (0-100%), length of elution=30 column volumes. The elutionprofile is shown in FIG. 14A and the protein gel is shown in FIG. 14B.Fractions A12-B7 were pooled (7 ml) and diluted ten times with startbuffer [0.02 M sodium phosphate buffer (pH 7.4), 0.5 M NaCl]. Thissample was loaded onto a second 1-ml HiTrap SP FF column (Pharmacia),repeating the same procedure as above, except that the elution wasperformed with a segmented gradient. The elution profile is shown inFIG. 15A and the protein gel is shown in FIG. 15B. Fractions A7-B12 werepooled (7 ml) and desalted as above using a 50-ml HiPrep 26/10 desaltingcolumn (Pharmacia). The protein concentration was measured by the methodof Bradford (Bradford, M. M., 1976, Anal. Biochem., 72, 248-254). Atotal of 41.61 mg of Arginase was purified from 760 ml cell culture. Theyield of purified Arginase was estimated to be 55.5 mg/l cell culture or1.3 mg/g wet cell weight.

Example 7 Comparison of Yield of Arginase Harvested and Purified UnderVarious Conditions

Table 2 below compares the yield of the Arginase produced under variousharvesting and purification conditions. These data show that harvestingcells 6 h after heat shock at a lower cell density of 12.8 produced ahigher Arginase yield of 132 mg/L after purification. TABLE 2 ArginaseYield (mg/L) Harvested 3 h after Harvested 6 h after Fed-batchFermentation heat shock heat shock Heat shock at OD 12.8 120 132 Heatshock at OD 25 — 55.5

Example 8A Preparation of the Pegylated Enzyme Using Cyanuric Chloride(CC) Activated Methoxypolyethylene Glycol

50 mg Arginase was dissolved in 20 ml PBS buffer solution (pH 7.4) to afinal concentration of 2.5 mg/ml. Heat activation of Arginase wascarried out at 60° C. for 10 minutes. After activation, the temperatureof the enzyme was allowed to bring back to room temperature. 1 gcyanuric chloride activated methoxypolyethylene glycol (mPEG-CC)(MW=5000, Sigma) was added to Arginase at mole ratio 1:140(Arginase:PEG). A magnetic stirring bar was used to stir the mixtureuntil all of the polyethylene glycol (PEG) was dissolved.

When all of the PEG was dissolved, pH of the PEG-Arginase mixture wasadjusted to 9.0 with 0.1 N NaOH, pH was further maintained at 9.0 forthe next 30 minutes with furter additions of NaOH. Pegylation wasstopped by adjusting pH back to 7.2 with addition of 0.1 N HCl.

The pegylated Arginase was dialyzed against 2-3 liters of PBS buffersolution, pH 7.4, at 4° C., with the use of a Hemoflow F40S capillarydialyzer (Fresenius Medical Care, Germany) to remove excess PEG. Afterdialysis, pegylated Arginase was recovered and the final concentrationwas readjusted.

The pegylated Arginase was filtered through a 0.2 pmn filter into asterilized container and was stored at 4° C. The ½-life of this enzymein a human patient was tested to be about 6 hours (see FIG. 21).

Example 8B Preparation of Arginase Expressed in B. subtilis and UsingEither CC or SPA at a Lower Peg Ratio

Pegylation was first developed by Davis, Abuchowski and colleagues(Davis, F. F. et al., 1978, Enzyme Eng. 4, 169-173) in the 1970s. Incontrast to modifying the formulation of a drug, chemical attachment ofpoly(ethylene glycol) PEG moieties to therapeutic proteins (a processknown as “pegylation”) represents a new approach that may enhanceimportant drug properties (Harris, J. M. et al., 2001, Clin.Pharmacokinet. 40, 539-551).

In 1979, Savoca et al. attached methoxypolyethylene glycol (mPEG) of5,000 Daltons covalently to bovine liver Arginase using2,4,6-trichloro-s-triazine (cyanuric chloride) as the coupling agent(Savoca, K. V. et al., 1979, Biochimica et Biophysica Acta 578, 47-53).The conjugate (PEG-Arginase) only retained 65% of its original enzymaticactivity. They reported that the blood-circulating life of PEG-Arginasein mice was extended over that of bovine Arginase. The half-life ofinjected bovine Arginase was less than 1 h, whereas that of thePEG-enzyme was 12 h. Their data also indicated that bovine Arginasemodified by PEG was rendered both non-immunogenic and non-antigenic whentested in mice.

Recombinant human Arginase (1.068 mg) was dissolved in 125 mM boratebuffer solution (pH 8.3) on ice or at room temperature. Activated mPEG,succinimide of mPEG propionic acid (mPEG-SPA; MW 5,000; ShearwaterCorporation) or mPEG activated with cyanuric chloride (mPEG-CC; MW5,000; Sigma), was added into the solution at Arginase:PEG mole ratiosof 1:50 or 1:20. This was performed in two stages. At the first stage,half of the PEG was added into the Arginase solution little by littleand mixed for 30 min gently on ice to prevent the pH getting out of therecommended range of 8.0-8.5. The other half of the PEG was added tothis solution and further mixed gently for 0.5-23 h. The mixture wasthen dialyzed against dH₂O by changing with dH₂O at least 3 times at 4degree C. using dialysis membrane with cut-off value of below 10,000.Both MnPEG-SPA and mPEG-CC use amino groups of lysines and theN-terminus of the protein as the site of modification.

When Arginase was modified on ice or at room temperature with MPEG-SPA(MW 5,000) using an Arginase:PEG mole ratio of 1:50, most of the enzymemolecules were modified after 1 h of reaction (FIG. 16). The sampleappeared the same even after 23 h of reaction. Arginase molecules wereattached with different numbers of PEG molecules and generated moleculesof various molecular weights. As expected, when a lower mole ratio of1:20 was used for the pegylation reaction, a higher proportion ofArginase was found in the non-pegylated form (FIG. 17). However, forboth of the mole ratios of Arginase:PEG used, longer reaction time andthe use of room temperature instead of ice did not seem to affect theextent of pegylation. With MPEG-SPA (MW 5,000), a mole ratio of 1:50 and1 h of reaction, the Arginase retained as much as 72-76% of its originalenzymatic activity (see Table 3 below), which is higher than thatreported for the bovine Arginase (65%; Savoca, K. V. et al., 1979,Biochimica et Biophysica Acta 578, 47-53).

When Arginase was modified on ice with mPEG-CC (MW 5,000) using anArginase:PEG mole ratio of 1:50, the reaction was quite slow and it took23 h to complete the pegylation (FIG. 18A). Moreover, most of the enzymemolecules were converted to a narrow spectrum of very high molecularweights. The reaction was much slower if a lower mole ratio of 1:20 wasused, as indicated in FIG. 18A.

Example 8C Preparation of Highly Active Pegylated Arginase

Fed-batch fermentation in a 15-L B. Braun Biostat C stainless steelfermentor was performed as described in Example 4. The cell culture (8.4L) collected at 4.5 h after heat shock at OD 12-13 was centrifuged at5,000 rpm for 20 min at 4 degree C. to pellet the cells. The culturesupernatant liquor was discarded and the cell pellet was stored at −80°C. The cells are stable at this temperature for a few days. To extractintracellular proteins, the cell pellet was resuspended in 1250 mlsolubilization buffer [50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 5 mM MnSO₄,lysozyme (75 μg/ml)]. After incubation at 30 degree C. for 20 min, themixture was divided into 300-ml portions in beakers, and each portionwas sonicated for 12 times, each time lasted for 10 s (the total timewas 120 s), at 2 min intervals using the Soniprep 150 Apparatus (MSE).About 5000 units of deoxyribonuclease I (Sigma D 4527) was added and themixture was incubated at 37 degree C. for 15 min to digest thechromosomal DNA. After centrifugation twice, each at 9,000 rpm for 30min at 4 degree C., the supernatant, containing the crude proteinextract, was assayed for the presence of the Arginase activity andanalyzed by SDS-PAGE (Laemmli, 1970, Nature, 227, 680-685).

The crude protein extract (1195 ml) was filtered and divided into 2portions, each contained 597.5 ml. Each portion was then loaded onto a130-ml Ni-NTA superflow (Qiagen) column (Pharmacia). Elution wasperformed with a linear gradient (0-100%) at a flow rate of 5 ml/minunder the following conditions: Buffer A=start buffer [0.02 M sodiumphosphate buffer (pH 7.4), 0.5 M NaCd]; Buffer B=start buffer containing0.5 M imidazole. Fractions containing pure Arginase were pooled andbuffer exchanged at 35 ml/min at 4 degree C. with PBS buffer, pH 7.4using the Pellicon XL device (polyether-sulphone membrane cut-off=8 kDa)and the lab-scale tangential flow filtration system (Millipore). Theprotein concentration was measured by the method of Bradford (Bradford,M. M., 1976, Anal. Biochem., 72, 248-254). A total of 788 mg of Arginasewas purified from 8.4 L cell culture. The yield of purified Arginase wasestimated to be 94 mg/l cell culture. The measured specific activity wasas high as 518 I.U./mg.

Pegylated Arginase with high specific activity was prepared in PBSbuffer. The purified Arginase (specific activity=518 I.U./mg) was in PBSbuffer before carrying out pegylation. The mPEG-SPA, MW 5,000 (5.82 g)was added into 555 ml of the purified Arginase (813.64 mg, 1.466 mg/ml)solution slowly in a 1-L beaker and then stirred for 2 h 40 min at roomtemperature (mole ratio of Arginase: rnPEG-SPA=1: 50). The mixture wasthen dialyzed extensively by ultra-dialysis against 15 L of PBS bufferusing the F50(S) capillary dialyser (Fresenius Medical Care) to removeall the unincorporated PEG. The mPEG-SPA uses amino groups of lysinesand the N-terminus of the protein as the site of modification. Themeasured specific activity of the pegylated Arginase is as high as 592I.U./mg. The results from SDS-PAGE analysis of the native and thepegylated Arginase are shown in FIG. 18B. The pegylated Arginase wasshown to be highly stable, in terms of Arginase activity and proteinconcentration, when stored in PBS buffer at 1 mg/ml for at least 3 weeksat room temperature. When stored at 4 degree C. in PBS buffer at 1mg/ml, it is stable for at least 6 months without decrease in specificactivity. TABLE 3 Activity (%) of Arginase when pegylated with variousactivated PEG at different mole ratios and temperatures. Activity (%) ofActivity (%) Arginase of Arginase (pegylated at Activity (%) of(pegylated at Activity (%) of Activity (%) Activity (%) of room Arginaseroom Arginase Arginase Arginase Time (h) temperature (pegylated ontemperature (pegylated on (pegylated on (pegylated on Allowed for withice with with ice with ice with ice with the Arginase:mPEG-Arginase:mPEG- Arginase:mPEG- Arginase:mPEG- Arginase:mPEG-Arginase:mPEG- Pegylation SPA SPA ratio of SPA SPA ratio of CC ratio ofCC ratio of Reaction ratio of 1:20) 1:20) ratio of 1:50) 1:50) 1:20)1:50) 0 100 100 100 100 100  100  1 83 76 76 72 ND ND 2 79 76 72 68 6864 5 83 74 74 72 65 65 23 75 72 72 64 66 66ND: Not determined.100% activity of Arginase is equivalent to 336 I.U./mg of protein.

Example 9A ½-Life Determination In Vivo of Arginase Obtained fromExample 8A

The pegylated Arginase was injected into a patient. A 3 ml blood samplein EDTA was taken from patient on a daily basis. The tube was pre-cooledto 4° C. on melting ice to prevent ex-vivo enzymatic reaction. The bloodwas then immediately spun down at 14000 rpm for 2 minutes to remove redblood cells. 1.5 ml supernatant (plasma) was pipetted out andtransferred to a new eppendorf tube. The plasma was then incubated at37° C. for 30 minutes. After incubation, arginine was added as asubstrate in concentration of 100 μM. Enzyme reaction was carried out at37° C. for 0, 10, 30, 60 minutes. At each time interval, reaction wasstopped by taking out 300 μl reaction sample to a new eppendorf tubecontaining 300 μl 10% trichloroacetic acid. Samples were taken and spunat maximum speed (14000 rpm) for 10 minutes. Supernatant was pipettedout and filtered with 0.45 μm filter. Finally, samples at different timeintervals were analysed with amino acid analyzer (Hitachi, L8800). Theresults are shown in FIG. 21.

Two batches of pegylated Arginase were prepared as described in Example8A during the studies. The first batch of pegylated Arginase wasprepared with Arginase:PEG mole ratio of 1:140. The second batch ofpegylated Arginase was prepared with Arginase:PEG mole ratio of 1:70.The pegylation protocol and condition used for preparing the two batcheswere identical (see Example 8A).

At time zero, 50 mg of the first batch of pegylated Arginase wasinfused. After 12 hours, another 50 mg of the first batch of pegylatedArginase was infused. The third Arginase infusion was done at hour 24during which another 50 mg of the first batch of pegylated Arginase wasused.

From hour 26 to hour 72, continuous infusion of the first batch ofpegylated Arginase (100 mg/day) was carried out instead of intermittentinfusion (50 mg/dosage). From hour 72 to hour 144, continuous infusionof the second batch of pegylated Arginase was carried out at a rate of100 mg/day. Continuous Arginase infusion was stopped at hour 144, andthe measurement of the half-life started from this point. The results ofthe half-life determination are shown in FIG. 22. Time zero in FIG. 22is equivalent to hour 144 in FIG. 21.

The results suggested that the half-life of the activity of the Arginasecould be divided into two phases. The first half-life of the pegylatedenzyme was about 6 hours. It took about 6 hours to reduce the relativeactivity from 100% to 50% (see FIG. 22). However, the second half-lifewas about 21 days. It took about 21 days to reduce the relative activityfrom 50% to 25%. This dual half-life effects might be due to a number offactors including the use of higher amount of mnPEG-CC in the pegylationand the specific infusion protocol used.

Example 9B ½-Life Determination of Pegylated Arginase In Vitro Using theMethod in Human Blood Plasma

Purified Arginase (1 mg) was dissolved in 1 ml of 125 mM borate buffersolution (pH 8.3) on ice. Activated PEG (mnPEG-SPA, MW 5,000) (7.14 mg)was added into the protein solution slowly at a mole ratio ofArginase:PEG=1:50. The mixture was stirred on ice for 2.5 h, followingthe method as described in Example 8B.

Pegylated Arginase (305.6 μl) at a concentration of 1 mg/ml was addedinto human plasma (1 ml) and the final concentration of pegylatedArginase was 0.24 mg/ml. The mixture was divided into 20 aliquots ineppendorf tubes (65 μl mixture in each eppendorf tube) and thenincubated at 37° C. A 1-2 μl portion of the mixture from each eppendorftube was used to test the Arginase activity. Results are shown in FIG.20. The ½-life was determined to be approximately 3 days. It took about3 days to reduce the relative activity from 100% to 50%. This isdetermined by using the curve in FIG. 20.

Example 10 Characterization of B. subtilis-Expressed Human Arginase andPegylated, Isolated and Purified Recombinant Human Arginase

(a) Measurement of Purity of Arginase by SDS-PAGE and Lumi-Imaging

Purified E. coli-expressed Arginase obtained from methods described byIkemoto et al. (Ikemoto et al., 1990, Biochem. J. 270, 697-703) wascompared to purified B. subtilis-expressed recombinant human Arginaseobtained from methods described in the present invention (FIGS. 19A and19B). Analysis of densities of total protein bands shown in FIG. 19Awith the Lumianalyst 32 program of Lumi-imager™ (Roche MolecularBiochemicals) indicated that the process developed in the presentinvention produced an Arginase that is to more than 99.9% pure (FIG.19B). However, an Arginase that is between 80-100% pure may also serveas the active ingredient to prepare a pharmaceutical composition. In thepreferred embodiment, recombinant Arginase of 80-100% purity is used. Inthe more preferred embodiment, the recombinant Arginase according to thepresent invention is 90-100% pure using SDS-PAGE followed bylumi-inaging.

(b) Measurement of Specific Activity by Coupled Reactions

The rate of the release of urea from 1-arginine by Arginase wasmonitored in a system containing urease, L-glutamate dehydrogenase andNADPH (Ozer, N., 1985, Biochem. Med. 33, 367-371). To prepare the mastermix, 0.605 g Tris, 0.0731 g a-ketoglutarate and 0.4355 g arginine weredissolved in 40 ml dH₂O. The pH was adjusted to 8.5 with 1 M HCl andthen 0.067 g urease was added to the mixture. The pH was furter adjustedto 8.3 with HCl before 0.0335 g glutamate dehydrogenase and 0.0125 gNADPH were added. The final volume was adjusted to 50 ml with dH₂O toform the master mix. The master mix (1 ml) was pipetted into a quartzcuvette. For measuring Arginase activity, 1-5 μg Arginase was added andthe decrease in absorbance at 340 nm (A₃₄₀) was followed for 1-3 min at30 degree C. One I.U. of Arginase was defined as the amount of enzymethat released 1 μmol of urea for 1 min under the given conditions. Thespecific activity of the purified recombinant human Arginase of thepresent invention was calculated to be 518 I.U./mg of protein, which wassignificantly higher than the reported values for purified humanerythrocyte Arginase (204 I.U./mg of protein; Ikemoto et al., 1989, Ann.Clin. Biochem. 26, 547-553) and the E. coli-expressed isolated andpurified recombinant human Arginase (389 I.U./mg of protein; Ikemoto etal., 1990, Biochem. J. 270, 697-703).

With mPEG-SPA (MW 5,000), a mole ratio of 1:50 and 2 h 40 min ofreaction, the pegylated Arginase retained as much as 114% of itsoriginal enzymatic activity (see Example 8C). That means the specificactivity of the pegylated human Arginase was 592 I.U./mg.

With MPEG-CC (MW 5,000), the pegylated human Arginase retained 64-68% ofits original enzymatic activity (Table 3), similar to that of thepegylated bovine Arginase (Savoca, K. V. et al., 1979, Biochimica etBiophysica Acta 578, 47-53,).

(c) Structural Characterization of the Native Arginase by ElectrosprayLC/MS

The B. subtilis-expressed and purified recombinant human Arginase,according to the amino aid sequence shown in FIG. 2B, contains 329 aminoacid residues at a theoretical molecular weight of 35,647.7 DaSimultaneous HPLC/UV and mass spectral analysis of the native Arginaseprovided a molecular weight of 35,634 Da. The observed molecular weightfor the native Arginase was found to correspond well with thetheoretical molecular weight of 35,647.7 Da derived from the expectedamino acid sequence of a 6×His-tagged human Arginase (FIG. 2B). Thepurity was found to be 98% by HPLC/UV based on LC/MS and 100% by LC/MSbased on HPLC/UV detection at 215 nr relative responses.

(d) Structural Characterization of the Native Arginase and the PegylatedArginase by Gel Filtration Chromatography

Studied by gel filtration chromatography in a HiLoad 16/60 superdex gelfiltration column (Pharmacia) at the protein concentration of about 2.8mg/ml in PBS buffer, the molecular weight of the native Arginase wasfound to be about 78 kDa and that of the pegylated Arginase (prepared inExample 8C) was about 688 kDa. As the molecular weight of monomericArginase is about 36 kDa, the results suggested that the native Arginaseexists as a dimer in PBS buffer.

(e) Secondary Structural Studies

Circular dichroism (CD) was used to analyze the secondary structures ofthe purified Arginases in a JASCO model J810 CD spectrometer. At equalprotein concentrations in 10 mM potassium phosphate buffer (pH 7.4), theCD spectrum of the native Arginase was found to be very similar to thatof the pegylated Arginase (prepared in Example 8C) when scanned from 195nm to 240 un, indicating that the native form and the pegylated form ofArginase have nearly identical secondary structures.

(f) Determination of VI Point

Using a Bio-Rad Model 111 mini IEF cell, the isoelectric point (pI) ofthe native Arginase (prepared in Example 8C) was found to be 9.0, whichis consistent with the published value of 9.1 in literature (Christopherand Wayne, 1996, Comp. Biochem. Physiol. 114B, 107-132).

(g) Functional Characterization and Determination of Kinetic Properties

Using the method reported by Ikemoto et al. (1990, Biochem. J. 270,697-703) for measuring Arginase activities, the native Arginase gave aK_(m) of 1.9±0.7 mM, a V_(max) of 518 μmol urea min⁻¹ mM⁻¹, a k_(cat) of2.0±0.5 s⁻¹, and a k_(cat)/K_(m) of 1.3±0.4 mM⁻¹ s⁻¹. The K_(m) value ofthe purified native Arginase was found to be similar to the publishedvalue (2.6 mM) of the human liver Arginase in literature (Carvajal, N.et al., 1999). Moreover, about 1 mM of Mn²⁺ ions and a temperature of30-50 degree C. are required to achieve maximum activity for the nativeArginase.

The pegylated Arginase gave a K_(m) of 2.9±0.3 nM and a V_(max) of 360μmol urea min⁻¹ mM⁻¹. The K_(m) value of the pegylated Arginase issimilar to that of the native Arginase, suggesting that the bindingaffinity towards arginine is retained after pegylation. Moreover, about1 mM of Mn²⁺ ions, a temperature of 40-50 degree C., and a pH of 10 arerequired to achieve maximum activity for the pegylated Arginase.

The functional properties of Arginase before and after pegylation aresimilar, which indicates that the covalent attachment of mPEG-SPAmolecules to Arginase improves its properties as a whole.

Example 11 Treatment Protocol Using Exogenously Adminstered Arginase

Blood samples of patients are taken daily throughout treatment forarginine levels, Arginase activities, complete blood picture and flillclotting profile. Renal and liver functions are taken at least everyother days, sooner if deemed necessary.

Vital signs (BP, Pulse, Respiratory rate, Oximeter reading) are takenevery 15 minutes for 1 hour after commencement of Arginase infusion thenhourly until stable. Thereafter, at the discretion of the treatingphysician.

20 minutes before Arginase infusion, premedication with dipheneramine 10mg iv. and hydrocortisone 100 mg iv. to be given before each freshinfusion of Arginase or at the discretion of the treating physician.

On day One Arginase is infuised over 30 minutes. Thereafter, Arginase isinfused weekly for at least 8 weeks. This may be continued ifanti-tumour activity is observed.

Example 12 Example of Treatment Protocol Using Exogenously AdministeredArginase

A 54-year old Chinese lady with metastatic rectal carcinoma withextensive pulmonary metastases that failed all standard treatments wastreated with pegylated recombinant Arginase in early August 2001. Hermain symptoms were cough, poor appetite and constipation. Her cancermarker CEA was 1100 U/ml. Informed consent for treatment with pegylatedrecombinant Arginase was obtained prior to treatment.

Treatment Methodology

850 mg of lyophilised recombinant Arginase I was administered. The drugwas reconstituted in PBS and pegylated. The pegylated enzyme was foundto be of fall activity.

Results

Results are shown in FIGS. 28 and 29. FIG. 28 shows satisfactorydepletion of arginine between 1-5 μM for 5 days (also see FIG. 21). FIG.29 shows a decrease of CEA levels from 1100 to 800 in 4 weeks.

1) Unlike chemotherapy this treatment resulted in no marrow suppressiveeffects or hair loss.

2) It can deplete arginine in a controlled manner, keeping the argininelevels within the therapeutic range (1-8 μM) for the desired period of 5days, which in vitro data suggest provide optimal tumour kill.

3) No major side effects and the patient tolerated the treatment withonly slight headache which may not be directly due to treatment.

4) Both biochemical and radiological improvement of the disease wasobserved after treatment, with CEA dropping by 30% and clearing up ofupper zone disease on chest X ray.

Example 13 In Vivo Arginne Depletion in Laboratory Rats with Arginase

In this example, four groups of rats (two in each group, one male andone female) were given dosages of various amounts of Arginase obtainedfrom example 8C on day 0. Blood samples were drawn from their tail veinson day 0 before intraperitoneal injection of the recombinant humanArginase, day 1 to day 6, then every 2 days.

As shown in FIG. 23, undetectable arginine level was achieved in allgroups and appeared to be dose dependent with 500 I.U. giving only afterone day of arginine depletion. With 1000 I.U. (500 I.U. administered inthe mormnig and another 500 I.U. administered in the afternoon), therewas a 4-day period with complete arginine depletion. With single dose of1500 I.U. administered, there was 6-day arginine depletion. By doublingthis dose to 3000 I.U., the duration of complete arginine depletion didnot appear to be prolonged to any further extent.

Therefore, 1500 I.U. of pegylated Arginase administeredintraperitoneally appears to be the optimal dose for arginine depletionwith undetectable arginine level for 6 days.

Example 14 Comparison of Changes in Level of Components in Blood BetweenNormal Rats and Rats with Zero Arginine Level Induced by Arginase fromDay 1 to Day 5

Intracardiac arterial blood samples were taken from a group of 5 rats onday 0 before administering Arginase. The day 0 samples served as theuntreated control. The level of total protein, albumin, globulin,SGOT/AST, SGPT/ALT, haemoglobin, fibrinogen A.P.T.T./second,prothrombin/second, number of white blood cells (WBC) and platelets weremeasured by Pathlab Medical Laboratory. Ltd, 2^(nd) Floor HenanBuilding, 90-92 Jaffe Road, Wanchai, Hong Kong. The rats then wereinjected with single dose of 1500 I.U. of Arginase intraperitoneally.Zero arginine level were achieved in all rats. From day 1 to day 5, onerat was sacrificed on each day and intracardiac arterial blood samplewas taken and measured by PathLab Medical Laboratory. Results show thatall proteins were within the normal ranges as cited by PathLab MedicalLaboratory.

Example 15 The Response Upon Arginine Depletion in Hep3B Tumour-BearingNude Mice

A human hepatoma cell line (Hep3B2.1-7) was inoculated subcutaneouslyinto the right flank of six BALB/c nude mice to induce growth of thetumour. Three randomly picked mice were administered intraperitoneallyonce a week with 500 I.U. pegylated Arginase obtained from the methoddescribed in example 8C while the other three mice were not given anyArginase treatment to serve as the control. The implanted mice wereobserved once every two days for the growth of the solid tumour in situby digital calliper measurements to determine tumour size which iscalculated according to the formula:Tumour size (mm)=average of two perpendicular diameters and one diagonaldiameter.The number of mice that died in each group was also recorded on a dailybasis.

As shown in FIG. 24, the rate of increase in size of the tumour per dayin control group was approximately 6 times the rate increase in grouptreated with pegylated Arginase for the first 20 days of the experiment.2 mice in the control group were dead within 24 days while the micetreated with pegylated Arginase can survive for at least 75 days.

Example 16 The Response Upon Arginine Depletion in PLC/PRF/5Tumour-Bearing Nude Mice

In this example, a solid tumour of human hepatoma (PLC/PRF/5) wasimplanted subcutaneously into the back of ten BALB/c nude mice to inducegrowth of tumour. Five randomly picked mice were administeredintraperitoneally once a week with 500 I.U. pegylated Arginase obtainedfrom the method described in example 8C while the other five mice weregiven 200 μl phosphate buffer saline (PBS) intraperitoneally to serve asthe control. The implanted mice were observed once every two days forthe growth of the solid tumour in situ by digital calliper measurementsto determine tumour size and mass. Tumour size is measured as describedin example 15 while tumour mass is calculated according to the formula:Tumour mass (mg)=length×width²/2 (assuming a specific gravity of 1.0g/cm³)(where length is the longest perpendicular diameter and width is theshortest perpendicular diameter)

As shown in FIG. 25A, the rate of increase in size of the tumour per daywas approximately 6.5 mm/day in the control group and the rate ofincrease in size of tumour in the group treated with pegylated Arginaseis approximately 5.3 mm/day for the first 39 days of the experiment. Asshown in FIG. 25B, the rate of increase in mass of the tumour per daywas approximately 1.8 times higher in the control group than that of thetreated group.

Example 17 The Response Upon Arginine Depletion in HuH-7 Tumour-BearingNude Mice

In this example, a solid tumour of human hepatoma (HuH-7) was implantedsubcutaneously into the back of ten BALB/c nude mice to induce growth oftumour. Five randomly picked mice were administered intraperitoneallyonce a week with 500 I.U. pegylated Arginase obtained from the methoddescribed in example 8C while the other five mice were given 200 μlphosphate buffer saline (PBS) per week intraperitoneally to serve as thecontrol. The implanted mice were observed once every two days for thegrowth of the solid tumour in situ by digital calliper measurements todetermine tumour size and mass as described in examples 15 and 16.

As shown in FIG. 26A, the rate increase in size of the tumour per daywas approximately 6.0 mm/day in control group and the rate of increasein size of tumour in the group treated with pegylated Arginase isapproximately 5.6 mm/day for the first 18 days of the experiment. Asshown in FIG. 26B, the rate of increase in mass of the tumour per daywas approximately 1.4 times higher in the control group than that of thetreated group.

Example 18 The Response Upon Arginine Depletion in MCF-7 Tumour-BearingNude Mice

In this example, a human breast cancer cell line (MCF-7) was inoculatedsubcutaneously into the right flank of four BALB/c nude mice to inducegrowth of tumour. Three randomly picked mice were administeredintraperitoneally once a week with 500 I.U. pegylated Arginase obtainedfrom the method described in Example 8C while the last one mouse werenot given any arginine treatment to serve as the control. The implantedmice were observed once every two days for the growth of the solidtumour in situ by digital calliper measurements to determine tumour sizeas described in Example 15.

As shown in FIG. 27, the tumour inoculated in the mice treated withpegylated Arginase disappeared within 20 days of the experiment

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural references unlessthe context clearly dictates otherwise. Thus, for example, reference to“a pharmaceutical preparation” includes mixtures of differentpreparations and reference to “the method of treatment” includesreference to equivalent steps and methods known to those skilled in theart and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to describe and disclose specificinformation for which the reference was cited in connection with. Theinvention having been fully described, modifications within its scopewill be apparent to those of ordinary skill in the art. All suchmodifications are within the scope of the invention.

Formulations of the pharmaceutical composition of the present inventioncan be used in the form of a solid, a solution, an emulsion, adispersion, a micelle, a liposome, and the like, wherein the resultingformulation contains one or more of the modified human arginase in thepractice of the present invention, as active ingredients, in a mixturewith an organic or inorganic carrier or excipient suitable for enteralor parenteral applications. The active ingredients may be the arginase,for example, with the usual non-toxic, pharmaceutically acceptablecarriers for tablets, pellets, capsules, suppositories, solutions,emulsions, suspensions, and any other form suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening and coloring agents andperfumes may be used. The active ingredients of one or more arginase areincluded in the pharmaceutical formulation in an amount sufficient toproduce the desired effect upon the target process, condition ordisease.

Pharmaceutical formulations containing the active-ingredientscontemplated herein may be in a form suitable for oral use, for example,as tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs. Formulations intended for oral use may be prepared according toany method known in the art for the manufacture of pharmaceuticalformulations. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract, thereby providing sustained action over a longerperiod. They may also be coated to form osmotic therapeutic tablets forcontrolled release.

In some cases, formulations for oral use may be in the form of hardgelatin capsules wherein the active ingredients are mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate,kaolin, or the like. They may also be in the form of soft gelatincapsules wherein the active ingredients are mixed with water or an oilmedium, for example, peanut oil, liquid paraffin, or olive oil.

The pharmaceutical formulations may also be in the form of a sterileinjectable solution or suspension. This suspension may be formulatedaccording to known methods using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1,4-butanediol. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed including synthetic mono- or diglycerides, fatty acids(including oleic acid), naturally occurring vegetable oils like sesameoil, coconut oil, peanut oil, cottonseed oil, or synthetic fattyvehicles, like ethyl oleate, or the like. Buffers, dextrose solutionspreservatives, antioxidants, and the like, can be incorporated or usedas solute to dissolve the soluble enzyme as required.

The pharmaceutical formulations may also be an adjunct treatmenttogether with other chemotherapeutic agents.

In the claims, an arginase that has an amino acid sequence substantiallythe same as the sequence shown in SEQ ID No. 9 means that the sequenceis at least 30% identical to that shown in SEQ ID No. 9 or that usingthe Arginase activity assay as described herein, there is no significantdifference in the enzymatic activity between the enzyme of SEQ ID No. 9and the one that is substantially similar. The six histidines areprovided for ease of purification, and the additional methionine groupprovided at the amino terminus thereof is to allow translation to beinitiated It is clear to one skilled in the art that other forms ofpurification may also be used, and therefore a “substantially similar”arginase does not need to have any homology with the MHH H sequence ofSEQ ID No. 3. In some bacterial strains there may be at least 40%homology with SEQ. SEQ ID No. 9. Some mammalian arginase may be 70%homology with SEQ ID No. 9.

1. An isolated recombinant human arginase I, comprising substantiallythe same amino acid sequence as set forth in SEQ ID NO: 9 and having apurity of 80-100%.
 2. The recombinant human arginase I according toclaim 1 further comprising six histidines attached to the amino terminalend thereof.
 3. The recombinant human arginase I according to claim 1having a specific activity of at least 250 I.U./mg.
 4. The recombinanthuman arginase I according to claim 3 having a specific activity of 500to 600 I.U./mg.
 5. The recombinant human arginase I according to claim4, comprising a modification that results in an in vitro plasmahalf-life of at least approximately 3 days.
 6. An isolated recombinanthuman arginase I according to claim 1, having a purity of at least 90%.7. The recombinant human arginase I according to claim 5, wherein saidmodification is pegylation.
 8. The recombinant human arginase Iaccording to claim 7, wherein said pegylation results from covalentlyattaching at least one polyethylene glycol (PEG) moiety to said arginaseusing a coupling agent.
 9. The recombinant human arginase I according toclaim 8, wherein said coupling agent is selected from the groupconsisting of 2,4,6-trichloro-s-triazine (cyanuric chloride, CC) andsuccinimide propionic acid (SPA).
 10. A method of producing recombinantprotein comprising: (a) cloning a gene encoding said protein; (b)constructing a recombinant Bacillus subtilis strain for expression ofsaid protein; (c) fermenting said recombinant Bacillus subtilis cellsusing fed-batch fermentation; (d) heat-shocking said recombinantBacillus subtilis cells to stimulate expression of said recombinantprotein; and (e) purifying said recombinant protein from the product ofsaid fermentation.
 11. The method according to claim 10 wherein saidBacillus subtilis is a prophage.
 12. The method according to claim 10wherein said protein is human arginase I.
 13. The method according toclaim 12 wherein said human arginase I comprises six histidines linkedto the amino-terminus thereof, and said purifying step comprisesaffinity chromatography in a chelating column.
 14. The method accordingto claim 12 wherein said fermenting step is performed using a feedingmedium consisting essentially of 180-320 g/L glucose, 2-4 g/LMgSO₄.7H₂O, 45-80 g/L tryptone, 7-12 g/L K₂HPO₄ and 3-6 g/L KH₂PO₄. 15.A pharmaceutical composition comprising an isolated and substantiallypurified arginase.
 16. The pharmaceutical composition according to claim15 wherein said recombinant human arginase is human arginase I.
 17. Thepharmaceutical composition according to claim 15 wherein saidrecombinant human arginase is human arginase I, further comprisingeentaifing six additional histidines attached to the amino terminal endthereof.
 18. The pharmaceutical composition according to claim 15,wherein said composition is further formulated in a pharmaceuticallyacceptable carrier.
 19. The pharmaceutical composition according toclaim 15, wherein the formulation of said pharmaceutical composition isin a form suitable for oral use, for a sterile injectable solution or asterile injectable suspension.
 20. The pharmaceutical compositionaccording to claim 16, wherein said recombinant human arginase I has aspecific enzyme activity of at least 250 I.U./mg.
 21. The pharmaceuticalcomposition according to claim 20, wherein said recombinant humanarginase I has a specific enzyme activity of 500 to 600 I.U./mg.
 22. Thepharmaceutical composition according to claim 16, wherein saidrecombinant human arginase I has a half-life in patient plasma of atleast 3 days.
 23. The pharmaceutical composition according to claim×22,wherein said recombinant human arginase I has a half-life in patientplasma of approximately at least 1 day.
 24. A method of treatment ofhuman malignancies, comprising administering human arginase I.
 25. Amethod of treatment of human malignancies comprising administering thepharmaceutical composition of claim
 15. 26. The use according to methodof claim 25, wherein said human malignancies are selected from the groupconsisting of: liver tumor, breast cancer, colon cancer and rectalcancer.
 27. A method of treatment of human malignancies comprisingadministering recombinant human arginase to a patient.
 28. A method oftreatment of human malignancies in a patient comprising administering apharmaceutical composition that reduces the physiological arginine levelin said patient to below 10 μM for at least 3 days.